LESSON 6: INFILTRATION AND EMBEDDING Flashcards
1
Q
✓ to fill all natural cavities, spaces & interstices of the tissues
A
IMPREGNATION
2
Q
✓ to give tissue samples a firm consistency
A
IMPREGNATION
3
Q
✓ impregnated tissue is placed into a precisely arranged position in a mold containing a medium which is then allowed to solidify
A
EMBEDDING
4
Q
mechanisms of solidification:
A
→crystallization
→evaporation of the solvent
→polymerization
5
Q
✓ Simplest, most common & best embedding medium
A
PARAFFIN PROCESSING
6
Q
Methods of Paraffin Impregnation
A
- MANUAL PROCESSING
- AUTOMATIC PROCESSING
- VACUUM EMBEDDING
7
Q
Substitutes for Paraffin Wax
A
- Paraplast
- Ester wax
- Water Soluble Waxes
8
Q
- Paraplast:
A
Embeddol
Bioloid
Tissue Mat
9
Q
EMBEDDING METHODS
A
WET METHOD
DRY METHOD
10
Q
TYPES OF PLASTIC MEDIA
A
- EPOXY 2. POLYESTER 3. ACRYLIC
11
Q
- EPOXY:
A
➢ Araldite base (bisphenol) - slowest
➢ Glycerol base (epon)
➢ Cyclohexene Dioxide (spurr) - fastest
12
Q
Types of Embedding Molds
A
- Leuckhart’s embedding mold
- Compound E unit
- Disposable Molds
- Plastic Embedding Rings & Base Molds
13
Q
Disposable Molds:
A
a. Peel-away (thin plastic Embedding molds)
b. Plastic Ice Trays
c. Paper boats
14
Q
➢ substance used to infiltrate, support and enclose tissue specimen
A
Infiltration/Embedding media
15
Q
➢ should be the same for infiltration and embedding
A
Infiltration/Embedding media
16
Q
Infiltration/Embedding media ➢ most important characteristic:
A
convertible from liquid to solid form
17
Q
✓ Simplest, most common & best embedding medium
A
PARAFFIN PROCESSING
18
Q
Clearing Agent Used
A
✓ xylene/benzene vs chloroform/cedarwood oil
19
Q
❖ 4 changes at 15-minute intervals
A
- MANUAL PROCESSING
20
Q
❖ 2-5 OC higher than the MP of the wax
A
- MANUAL PROCESSING
21
Q
❖ 2 – 3 changes with agitation
A
- AUTOMATIC PROCESSING
22
Q
❖ At least 3 OC higher than the MP of the wax
A
- AUTOMATIC PROCESSING
23
Q
❖ negative atmospheric pressure (400-500 mmHg)
A
- VACUUM EMBEDDING
24
Q
❖ Heat & vacuum
A
- VACUUM EMBEDDING
25
❖ 3 changes
3. VACUUM EMBEDDING
26
❖ 2 - 4 OC above MP of wax
3. VACUUM EMBEDDING
27
❖ ADV: Effects of heat are prevented
3. VACUUM EMBEDDING
28
❖ Applications in the lab: urgent biopsies, dense and fibrous tissues, delicate tissues
3. VACUUM EMBEDDING
29
➢ Fresh was should be filtered at
2 OC above its MP
30
➢ Wax from trimmings should be filtered with a
course filter paper
31
➢ Water is removed by heating the wax to
100-105 OC
32
✓ Paraffin wax should be
pure
33
Paraffin wax may be utilized
twice
34
✓ Melted paraffin: (?)above the MP of wax
✓ Cooling ➢ Ref at (?)
➢ immerse in cold water
➢ Tissue Tek with cold plate
5-10 OC
-5 OC
35
➢ MP: 56-57 OC
Paraplast
36
➢ More elastic & resilient
Paraplast
37
➢ Doesn’t require cooling
Paraplast
38
➢ MP= 56 – 58 OC
❖ Embeddol
39
➢ Less brittle than paraplast
❖ Embeddol
40
➢ Semisynthetic wax that is used in impregnating and embedding eye samples
❖ Bioloid
41
➢ Product of paraffin that contains rubber
❖ Tissue Mat
42
➢ MP: 46 – 48 OC
Ester wax
43
➢ Lower melting point but harder than paraffin
Ester wax
44
➢ Soluble in 95% EA & other clearing agents
Ester wax
45
Ester wax ➢ PROCESSING:
cellosolve/xylene → equal parts clearing agent + ester wax for 36 hours → 3-4 changes of pure ester wax → embedding → sectioning using heavy duty microtome
46
➢ Polyethylene glycols
Water Soluble Waxes
47
➢ Does not require dehydration & clearing
Carbowax
48
Fixation → wash-out → carbowax processing
Carbowax
49
✓ Reduces the processing time
Carbowax
50
➢ Does not remove neutral fats & lipids
Carbowax
51
➢ For histochemical and enzyme studies
Carbowax
52
➢ 70 % (?) → 90% (?) → 100% (2 changes for [?]) at 56 OC
30 min
45 min
1 hr each
53
➢ Blocking at 50 OC →rapidly cooled at ref temp
Carbowax
54
➢ Applications: histochemical and enzyme studies; for the demonstration of neutral fats and lipids
Carbowax
55
➢ DISADVANTAGE: hygroscopic reagent
Carbowax
56
Carbowax ➢ REMEDY: → Add (?) in waterbath → Use floating solutions: (?)
soap or 10% polyethylene glycol 900
Pearse solution or Blank and McCarthy solution
57
✓ Purified pyroxylin nitrocellulose
CELLOIDIN PROCESSING
58
✓ Suitable for specimens containing large cavities or hollow spaces which tend to collapse (eyes) & for larger embryos
CELLOIDIN PROCESSING
59
✓ Available in thin (2%), medium (4%) and thick (8%) solutions
CELLOIDIN PROCESSING
60
✓ DIS: Tissues cannot be cut as thin as they are with paraffin wax
CELLOIDIN PROCESSING
61
✓ ADV: Causes much less shrinkage & distortion; slow process
CELLOIDIN PROCESSING
62
➢ With rubbery consistency – w/o distortion
CELLOIDIN PROCESSING
63
➢ Does not require heat
CELLOIDIN PROCESSING
64
➢ Soluble in equal concentrations of ether and alcohol
Low Viscosity Nitrocellulose
65
✓ Can be used in higher concentrations
Low Viscosity Nitrocellulose
66
✓ Produces harder tissue blocks
Low Viscosity Nitrocellulose
67
o Cracking of tissue sections and chrome-mordanted tissues may crumble
Low Viscosity Nitrocellulose
68
→ REMEDY: add plasticizers like Castor oil, Oleum ricini
Low Viscosity Nitrocellulose m
69
o More explosive than celloidin
Low Viscosity Nitrocellulose
70
o Cannot be stored
Low Viscosity Nitrocellulose
71
❖ for bones, teeth large brain sections & whole organs
WET METHOD
72
WET METHOD ❖ Fixation → Dehydration → Equal parts ether and alcohol (12-24 hours) → Thin celloidin: 2-4 % (5-7 days) → Medium celloidin: 4-6 % (5-7 days) → Thick celloidin: 812 % (3-5 days) → Embedding: fresh thick celloidin in a jar or desiccator (fingerprint no longer leaves a mark on block surface) → Storage: 70-80% alcohol
73
❖ Same with wet method EXCEPT for the following steps
DRY METHOD
74
✓ Gilson’s mixture (chloroform + cedarwood oil) is added to the fresh thick celloidin during embedding
DRY METHOD
75
✓ Storage in alcohol is contraindicated
DRY METHOD
76
❖ Provided superior results for light microscopic studies especially of hard tissues and samples for high resolution microscopy
PLASTICS
77
- slowest
Araldite base (bisphenol)
78
- fastest
Cyclohexene Dioxide (spurr)
79
➢ Reduce antigenicity
EPOXY
80
➢ Sensitization on skin contact and inhalation
EPOXY
81
➢ Contains toxic components (eg. Vinylcyclohexane dioxide)
EPOXY
82
➢ For high resolution LM
ACRYLIC
83
: valued for its hydrophilic nature
➢ Glycol Methacrylate (GMA)
84
: valued for its hardness
➢ Methyl Methacrylate (MMA)
85
– added as catalyst
➢ Benzoyl Peroxide
86
→ acts as an active site (polymerization of acrylics)
➢ Benzoyl Peroxide
87
✓ Histochemical and enzyme studies
GELATIN PROCESSING
88
✓ Delicate specimens
GELATIN PROCESSING
89
✓ Frozen sections
GELATIN PROCESSING
90
✓ Water-soluble
GELATIN PROCESSING
91
✓ Low MP and does not overharden
GELATIN PROCESSING
92
GELATIN PROCESSING ❖ Fixation → washing → 10 % (24 hours) → 20 % (12 hours) → 20% until impregnation and embedding are complete → 10 % formalin (12-24 hours)
93
**All gelatin reagents contain
1 % phenol
94
✓ Arranging the tissue in the mold:
embedding
95
✓ Fixing the tissue block on the microtome:
prior to sectioning
96
✓ Fixing the tissue block on the microtome:
prior to sectioning
97
✓ Arranging the tissue ribbons on the slide:
flotation
98
✓ 2 L-shaped heavy metal arranged flat on a metal surface
1. Leuckhart’s embedding mold
99
✓ with several interlocking plates making several compartments
2. Compound E unit
100
✓ ADV: cheap, diff sizes, avoids confusion
Paper boats
